Unsustainable demands, rising oil costs and concerns over climate change have inspired interest in renewable fuels and chemicals (Fortman et al., 2008). Microbial production of high-energy fuels via economically efficient and environmentally sustainable bioprocesses has recently emerged as a viable alternative to the conventional production of transportation fuels (Lynd et al., 2005). Fatty acids, sometimes touted as nature's ‘petroleum’, are long-chain carboxylic acids that cells use for both chemical and energy storage functions. These energy-rich molecules are currently derived from plant oils and animal fats. However, increasing food prices worldwide have rekindled debate over the competition of agricultural resources between the energy sector and the food industry. Therefore, alternatives to agricultural crops are urgently needed for the production of sustainable and economical biofuels. Namely, producing fatty acid-derived biofuels directly from abundant and cost-effective renewable resources by microbial fermentation is an attractive alternative biofuel production method.
In the phospholipid form, fatty acids are a major component of cell membranes in all organisms. Certain species of yeasts and microalgae can accumulate fatty acids in the neutral form as triacylglycerols (TAG) at up to 30-70% of dry cell weight (Beopoulos et al., 2009). While naturally possessing a lower lipid content (between 3.5 and 10.7% of DCW) (Johnson et al., 1972), S. cerevisiae offers several advantages over oleaginous yeasts and microalgae as a production host for fatty acids and derivatives. Namely, S. cerevisiae is more genetically tractable than oleaginous yeasts and microalgae; thus, genetic tools for metabolic pathway manipulation are more abundant. Second, the generation, isolation, and analysis of S. cerevisiae mutant strains can be performed with relative ease, and deletion strains for most coding genes are commercially available. Third, S. cerevisiae has a proven track record in various industrial applications, and the fermentation of S. cerevisiae has been previously manipulated to produce numerous heterologous metabolites. Finally, S. cerevisiae is easily cultivated in chemically defined medium and exhibits fast growth rates, thus facilitating scaling-up processes.
Because fatty acids are integral parts of all living organisms, their biosynthesis and regulation have been comprehensively studied in both prokaryotes and eukaryotes (Magnuson et al., 1993; Tehlivets et al., 2007). In the yeast S. cerevisiae, fatty acid biosynthesis serves many important functions including energy metabolism, posttranslational protein modifications and membrane lipid biosynthesis. De novo fatty acid biosynthesis in S. cerevisiae requires acetyl-CoA carboxylase (ACC; encoded by the ACC1 gene) and the fatty acid synthase complex (FAS; encoded by FAS1 and FAS2) (Al-Feel et al., 1992) (FIG. 1). ACC converts acetyl-CoA into malonyl-CoA. Subsequently, the FAS complex condenses one equivalent of acetyl-CoA and 7-8 equivalents of malonyl-CoA into C16-C18 fatty acyl-CoAs. Yeast FAS complex is a 2.6-MDa protein consisting of two non-identical, multifunctional subunits, α and β, organized as a hexamer (α6β6) (Schweizer and Hofmann, 2004). The α subunit, encoded by FAS2, contains β-ketoacyl synthase (KS), β-ketoacyl reductase (KR), and acyl carrier protein (ACP) domains. The β subunit, encoded by FAS1, contains acetyl-, malonyl-, and palmitoyl-transferase (AT and MPT), as well as dehydratase (DH) and enoyl reductase (ER) domains. As they emerge from the FAS complex, newly synthesized fatty-acyl CoAs are bound to acyl-CoA binding protein (ACBP; encoded by the ACBP1 gene), which facilitates intracellular transport of acyl-CoA to the endoplasmic reticulum and lipid bodies for phospholipids and TAG biosynthesis (Knudsen et al., 1999). Notably, all of S. cerevisiae C16-C18 fatty acid biosynthesis enzymes are encoded by merely two genes (FAS1 and FAS2), as opposed to ten separate genes (FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, Acp and TesA) as is the case for E. coli. This distinction allows us to overexpress the entire pathway in a more straightforward manner.
Because fatty acids serve multiple cellular functions in yeast, their biosynthesis—from the conversion of acetyl-CoA to malonyl-CoA by ACC to the subsequent production of fatty acyl-CoA by the FAS complex—is tightly regulated at multiple levels (Tehlivets et al., 2007). Moreover, fatty acid biosynthesis is feedback inhibited by long chain acyl-CoA. ACC is inhibited by extremely low concentrations of long-chain acyl-CoA, (Ki=1-5 nM) (Ogiwara et al., 1978). Altogether, these mechanisms ensure that the cell does not accumulate excess quantities of this energy-rich metabolite. In order to overproduce fatty acid-derived biofuels in S. cerevisiae, these regulatory elements must be mitigated. A common strategy to relieve feedback inhibition by acyl-CoA is the overexpression of either the endogenous or heterologous acyl-acyl carrier protein (ACP) or acyl-CoA thioesterase to produce free fatty acids (FIG. 1).
While TAGs and free fatty acids are valuable, they cannot be used directly as fuels and must first be chemically processed prior to utilization. Therefore, renewable fuels that are directly compatible with existing infrastructure are in great demand. Over 1 billion gallons of biodiesel, a renewable alternative to diesel fuel, are produced each year in the US alone (US_Environmental_Protection_Agency). Composed of fatty acid methyl and ethyl esters (FAMEs and FAEEs, respectively), biodiesel is traditionally derived from the chemical transesterification of plant oils and animal fats (Hill et al., 2006). Fatty alcohols are also important oleochemicals and find many industrial applications ranging from lubricants to cosmetics. Traditionally, fatty alcohols are produced in two chemical steps from plant oils and animal fats: 1) transesterification/hydrolysis of plant oils and animal fats to methyl esters and fatty acids and 2) hydrogenation of methyl esters and fatty acids to fatty alcohols.